Diagnostic method for determination of vanilmandelic acid in urine



United States Patent U.S. Cl. 23-230 5 Claims ABSTRACT OF THE DISCLOSURE Qualitative and quantitative determinaion of vanilmandelic acid in urine via chromatographic means.

This invention relates to a novel means for diagnosing a pathological condition. More particularly, it is concerned with a diagnostic method for delermining abnormal amounts of vanilamandelic acid in the urine of humans.

Recently, it has been shown that an excessive amount of vanilmandelic acid (VMA) in a urine specimen strongly suggests the presence of certain tumors, namely, pheochromocytoma and neuroblastoma. It is the practice generally, however, not to perform any tests to ascertain the presence of VMA as an indication of the above tumors unless the patient indicates definite symptoms, the reason being that the test commonly used was costly and timeconsuming. One symptom which is generally observed with the above tumors, although not conclusive, is hypertension. The aforesaid commonly used test consists of a colorimetric method which analyzes for vanillin a degradation product of VMA. With the discovery of the present diagnostic method, due to its inexpensiveness and shorter time period, a routine screen is now available for all patients who are hypertensive and, hence, can be included in the general series of tests usually administered to hypertensive patients.

Accordingly, the present invention relates to a diagnostic method for determining vanilmandelic acid in suspect urine which comprises:

(a) Commingling a sample of acidified suspect urine with a non-miscible organic solvent, removing an aliquot from said organic phase, evaporating therefrom all solvent and treating the residue with a minimal amount of a reconstituting solvent which dissolves the vanilmandelic acid in said residue;

(b) Spotting the resulting solution on an absorbent carrier;

(c) Developing chromatographically the non-volatile components of said solution by contacting said absorbent carrier with a chromatographic developer consisting of from about 60 to about 80 volume percent of an alcohol in aqueous solution; and

(d) Drying said absorbent carrier in the air and treating said carrier with a color developer whereby the presence of vanilmandelic acid in said urine is established by the formation of a characteristic colored zone.

In step (a) above, the sample can be acidified with any acid organic or inorganic, however, in any event it is generally preferred to make the resulting solution strongly acidic, i.e., pH 3. For purposes of this invention, hydrochloric acid is most preferred. The term non-miscible organic solvent can include any organic solvent'which is not soluble in water to any appreciable extent. For the most part, it is preferred to use a solvent which is polar in nature; although, solvents such as benzene, toluene and xylene will provide adequate results. In this regard, adequate results are obtained when there is sufficient partition of vanilmandelic acid between the two 3,482,942 Patented Dec. 9, 1969 phases. A particularly effective solvent is ethyl acetate and in most cases will be the solvent of choice.

The reconstituting solvent can be any solvent which substantially dissolves all of the VMA present in the residue. Generally, the reconstituting solvent will be the same as the non-miscible organic solvent utilized in the experiment although it need not be. In fact, the reconstituting solvent can be water and in some instances is preferred.

In connection with the evaporation and reconstitution steps, it is possible to regulate the evaporation carefully so that a small amount of solvent remains. If accomplished, there would be no need to reconstitute; however, such a regulation is extremely difficult and on a physical manipulation basis, it is easier to evaporate completely and subsequently add the desired amount of solvent.

With regard to step (b), the terms employed herein and in the claims are well known to those skilled in the art of chromatography. By the term spotting is meant the technique of applying to an absorbent carrier a minute drop of a liquid which contains non-volatile components to be separated by chromatography. Ordinarily, the volatile components of the liquid sample are evaporated before development of the chromatogram. By absorbent carrier, it is meant to contemplate a material which can, for example, be made of cellulose, polyvinyl chloride, or from modified forms of cellulose or even from glass fibers, although it is preferred to use paper made from cellulose.

By the term developing chromatographically used in step (c) above, is meant the technique of separating the non-volatile substituents of the mixture spotted on the carrier by causing a liquid phase to move through the absorbent carrier during which separation of the nonvolatiles takes place. In the practice of this method, the movement of the developer front edge may be downwardly (descending paper chromatography), upwardly (ascending paper chromatography), or even horizontally (circular, disk or radial paper chromatography). It is also possible, although less desirable, to use the technique of thin layer chromatography. It is, however, especially convenient for less skilled technicians to use the ascending paper chromatography method for purposes of this invention.

The non-volatile components of the liquid sample residue spotted on the absorbent carrier is developed chromatographically with a chromatographic developer solvent system which for the present invention consists of from about 60 to about volume percent of an alcohol in aqueous solution. Said volume percent limitation will necessarily restrict the type of alcohol which can be utilized. In general, aliphatic alcohols containing from 1 to 6 carbon atoms or mixtures thereof will be suitable. This would include, for example, alcohols such as methanol, ethanol, n-propanol, isopropanol, isobutanol, nbutanol, Z-butanol and t-butanol. Of course, other alcohols may work, however, they must meet the requirement of having the ability of being soluble in water to the extent of forming 60 to 80 volume percent solutions. This requirement will necessarily exclude many alcohols.

The absorbent carrier, e.g., strips, are placed in the chromatography jar containing said chromatographic developer and the chromatogram is allowed to develop until the solvent front reaches to Within one-half inch of the top of the strip. After chromatography is complete, the strips or absorbent carriers are removed from the jars and allowed to dry (step (d) above). When dried, the strips are sprayed with a color developer establishing the presence of vanilmandelic acid by the formation of a characteristic color zone. For purposes of this invention, said color developer is a diazonium salt and the color results from the reaction between a diazonium salt and a phenolic compund. Since the rate of reaction depends upon pH wherein the optimum is pH 8-9, the strips are subsequently sprayed with an aqueous solution containing any suitable base. For t p (d) above, any stable diazonium salt will work, for example, diazotized p-nitroaniline, diazotized sulfanilic acid and tetrazatized benidine. As for the basic solutions mentioned above, suitable examples include aqueous potassium carbonate, aqueous sodium hydroxide and aqueous potassium hydroxide solutions.

It is advantageous, in carrying out the process of this invention, to use paper as the absorbent carrier in strip form and to use as chromatographic developer a mixture consisting of 80% t-butanol and 20% distilled water on a volume basis. The paper may be in the form of small or large strips or small or large sheets, but it is especially convenient to use strips that are about one-inch wide by five inches long. The type of paper employed is not critical to this invention. However, for best results, only the most highly purified analytical grades of filter paper ought to be used. These are widely available as filter paper, chromatographic grades, from laboratory supply houses, worldwide. It has been found that especially effective results can be obtained if the paper used is that known by the tradename and number, Whatman No. 1, which is manufactured by W. & R. Balston Ltd., and which may be obtained in the United States from H. Reeve Angel & Co., Inc., 52 Duane St., New York 17, NY.

During the chromatographic development, the system should be kept in an area free from drafts and subject to minimum fluctuations in temperature. Resolution appears to be best when the chromatography is carried out at room temperature, however, adequate resolutions can be obtained at various temperatures up to and including 50 C. provided there is a minimum of temperature fluctuation. Once the strip is removed, dried and developed, the presence of vanilmandelic acid is ascertained not only by the characteristic color but also by the typical Rf value. Rf is the ratio of the distance travelled from the origin of the spot to that travelled by the solvent front. For any constant concentration of VMA, and solvent system, the Rf value will always be the same. Experimentally, it is observed that vanilmandelic acid is the slowest moving component of those present in the extract as obtained herein regardless of the solvent system used.

The following examples are given by way of illustration and should not be interpreted as limiting the invention, the scope of which is defined by the appended claims.

EXAMPLE I A urine sample (9 ml.) is made strongly acid by the addition of concentrated hydrochloric acid (final pH=l). An equal volume of ethyl acetate is added thereto and the mixture is vigorously mixed. The mixture is then allowed to separate into two layers whereupon a ml. aliquot of the ethyl acetate layer is removed and evaporated to dryness. Said evaporation is achieved with the aid of a Warm water bath and a stream of nitrogen. The resulting residue is reconstituted with 0.5 ml. of ethyl acetate and a small amount of this solution is spotted on a strip (1 x 5 inches in size) of Whatman No. 1 chromatography grade filter paper. In the adjacent area is placed a spot from a positive control containing vanilmandelic acid (10 mg/1.).

The strip is then placed in an 8 ounce wide-mouth jar fitted with a screw cap and a downwardly-suspended hook (on which the paper is hung) containing 10 ml. of an 80-20 volume percent mixture of t-butanol and water. The chromatogram is then allowed to develop until the solvent front reaches to within approximately 0.5 inch of the top of the strip. The strip is removed and allowed to air-dry whereupon the strip is sprayed with a freshly prepared 0.2% aqueous solution of diazotized p-nitroaniline. The strip is air-dried once again and then sprayed with an 0.4 M aqueous sodium carbonate solution. The purple spot with a Rf of approximately 0.3 is due to VMA. If this spot is more intense than the spot due to the control, the Sample contains a concentration of VMA greater than 10 mg./l. If the spot is less intense than the control but still visible, the sample contains a concentration of VMA between 5 mg./l. and 10 mg./l. If no purple spot is visible on the sample side, the sample contains less than 5 mg./l. of VMA.

EXAMPLE II The procedure of Example I is repeated wherein the following non-miscible organic solvents are used in place of ethyl acetate with comparable results:

benzene toluene xylene methyl ethyl ketone n-butyl acetate EXAMPLE III The procedure of Example I is repeated wherein the following chromatographic developers are used in lieu of an -20 volume percent mixture t-butanol and water with comparable results:

60-40 volume percent of t-butanol and water, respectively.

60-40 volume percent of n-butanol and water, respectively.

70-30 volume percent of ethanol and water, respectively.

80-20 volume percent of isopropanol and water, respectively.

70-30 volume percent of benzyl alcohol and water, re

spectively.

EXAMPLE IV The procedure of Example I is repeated wherein the following diazonium salts are used in place of diazotized p-nitroaniline with comparable results:

diazotized sulfanilic acid tetrazotized benzidine What is claimed is:

1. A diagnostic method for determining vanilmandelic acid in urine which comprises:

(a) commingling a sample of acidified urine with a non-miscible organic solvent, removing an aliquot from said organic phase, evaporating therefrom all solvent and treating the residue with a minimal amount of a reconstituting solvent which dissolves the vanilmandelic acid in said residue;

(b) spotting the resulting solution on an absorbent carrier;

(0). developing chromatographically the non-volatile components of said solution by contacting said absorbent carrier with a chromatographic developer consisting of from about 60 to about 80 volume percent of an alcohol in aqueous solution; and

(d) drying said absorbent carrier in the air and treating' said carrier with a color developer whereby the presence of vanilmandelic acid in said urine is established by the formation of a characteristic colored zone.

2. The method of claim 1 wherein said non-miscible organic solvent is ethyl acetate.

3. The method of claim 1 wherein said chromatographic developer is an 80-20 volume percent mixture containing t-butanol and water, respectively.

4. The method of claim 1 wherein said color developer is a diazonium salt subsequently made basic in order to allow the formation of said colored zone.

5. A diagnostic method for determining vanilmandelic acid in urine which comprises:

(a) commingling from about 8 to about 10 ml. of an acidified urine sample with an equal volume of ethyl acetate, removing a 5 ml. aliquot of the ethyl acetate layer, evaporating therefrom all solvent and treating the residue with 0.5 ml. of ethyl acetate;

(b) spotting the resulting solution on an absorbent carrier;

(0) developing chromatographically the non-volatile components of said solution by contacting said absorbent carrier with a chromatographic developer consisting of an 80-20 volume percent mixture of t-butanol and water respectively; and

(d) drying said absorbent carrier in the air and treat- 15 References Cited Armstrong, M. D., et al., I. of Biol. Chem, N01. 218, pp. 293-303 (1956).

Hill, G. A. et al., chemistry and industry, 1959, p. 399

MORRIS O. WOLK, Primary Examiner ELLIOTT A. KATZ, Assistant Examiner 

